Comparative Transcriptome Analysis Points to the Biological Processes of Hybrid Incompatibility between Brassica napus and B. oleracea.

Improving Brassica napus via introgression of the genome components from its parental species, B. oleracea and B. rapa, is an important breeding strategy. Interspecific hybridization between B. napus and B. rapa is compatible with high rate of survival ovules, while the hybridization between B. napus and B. oleracea is incompatible with the high occurrence of embryo abortion. To understand the diverse embryo fate in the two interspecific hybridizations, here, the siliques of B. napus pollinated with B. oleracea (AE) and B. rapa (NE) were employed for transcriptome sequencing at 8 and 16 days after pollination. Compared to NE and the parental line of B. napus, more specific differentially expressed genes (DEGs) (1274 and 1698) were obtained in AE and the parental line of B. napus at 8 and 16 days after pollination (DAP). These numbers were 51 and 5.8 times higher than the number of specific DEGs in NE and parental line of B. napus at 8 and 16 DAP, respectively, suggesting more complex transcriptional changes in AE. Most of DEGs in the terms of cell growth and cell wall formation exhibited down-regulated expression patterns (96(down)/131(all) in AE8, 174(down)/235(all) in AE16), while most of DEGs in the processes of photosynthesis, photorespiration, peroxisome, oxidative stress, and systemic acquired resistance exhibited up-regulated expression patterns (222(up)/304(all) in AE8, 214(up)/287(all) in AE16). This is in accordance with a high level of reactive oxygen species (ROS) in the siliques of B. napus pollinated with B. oleracea. Our data suggest that the disorder of plant hormone metabolism, retardation of cell morphogenesis, and the accumulation of ROS may be associated with hybrid incompatibility between B. napus and B. oleracea.

Genetic analysis and QTL mapping for silique density in rapeseed (Brassica napus L.).

KEY MESSAGE: Genetic models, QTLs and candidate gene for silique density on main inflorescence of rapeseed were identified. Silique density is one of the critical factors to determine seed yield and plant architecture in rapeseed (Brassica napus L.); however, the genetic control of this trait is largely unknown. In this study, the genetic model for silique density on main inflorescence (SDMI) of rapeseed was estimated according to the phenotypic data of P1 (an inbreed line with high SDMI), P2 (an inbreed line with low SDMI), F1, F2, BC1P1 and BC1P2 populations, revealing that SDMI is probably controlled by multi-minor genes with or without major gene. The QTLs for SDMI and its component characters including silique number on main inflorescence (SNMI) and main inflorescence length (MIL) were consequently mapped from a DH population derived from P1 and P2 by using a genetic linkage map constructed by restriction site-associated DNA sequencing (RAD seq) technology. A total of eight, 14 and three QTLs were identified for SDMI, SNMI and MIL under three environments, respectively, with an overlap among SDMI and SNMI in 55.7-75.4 cm on linkage group C06 which corresponding to 11.6-27.3 Mb on chromosome C06. Genomic resequencing was further conducted between a high- and a low-SDMI pool constructed from the DH population, and QTL-seq analysis identified a 0.15 Mb interval (25.98-26.13 Mb) from the C06-QTL region aforementioned. Transcriptome sequencing and qRT-PCR identified one possible candidate gene (BnARGOS) from the 0.15 Mb interval. This study will provide novel insights into the genetic basis of SD in rapeseed.

Selective modes affect gene feature and function differentiation of tetraploid Brassica species in their evolution and domestication.

The genus Brassica contains a diverse group of important vegetables and oilseed crops. Genome sequencing has been completed for the six species (B. rapa, B. oleracea, B. nigra, B. carinata, B. napus, and B. juncea) in U's triangle model. The purpose of the study is to investigate whether positively and negatively selected genes (PSGs and NSGs) affect gene feature and function differentiation of Brassica tetraploids in their evolution and domestication. A total of 9,701 PSGs were found in the A, B and C subgenomes of the three tetraploids, of which, a higher number of PSGs were identified in the C subgenome as comparing to the A and B subgenomes. The PSGs of the three tetraploids had more tandem duplicated genes, higher single copy, lower multi-copy, shorter exon length and fewer exon number than the NSGs, suggesting that the selective modes affected the gene feature of Brassica tetraploids. The PSGs of all the three tetraploids enriched in a few common KEGG pathways relating to environmental adaption (such as Phenylpropanoid biosynthesis, Riboflavin metabolism, Isoflavonoid biosynthesis, Plant-pathogen interaction and Tropane, piperidine and pyridine alkaloid biosynthesis) and reproduction (Homologous recombination). Whereas, the NSGs of the three tetraploids significantly enriched in dozens of biologic processes and pathways without clear relationships with evolution. Moreover, the PSGs of B. carinata were found specifically enriched in lipid biosynthesis and metabolism which possibly contributed to the domestication of B. carinata as an oil crop. Our data suggest that selective modes affected the gene feature of Brassica tetraploids, and PSGs contributed in not only the evolution but also the domestication of Brassica tetraploids.

Identification of Candidate Genes for Clubroot-Resistance in Brassica oleracea Using Quantitative Trait Loci-Sequencing.

Clubroot caused by Plasmodiophora brassicae is a devastating disease of cabbage (Brassica oleracea). To identify quantitative trait loci (QTLs) for clubroot resistance (CR) in B. oleracea, genomic resequencing was carried out in two sets of extreme pools, group I and group II, which were constructed separately from 110 and 74 F2 cloned lines derived from the cross between clubroot-resistant (R) cabbage "GZ87" (against race 4) and susceptible (S) cabbage "263." Based on the QTL-sequencing (QTL-Seq) analysis of group I and group II, three QTLs (i.e., qCRc7-2, qCRc7-3, and qCRc7-4) were determined on the C07 chromosome. RNA-Seq and qRT-PCR were conducted in the extreme pools of group II before and after inoculation, and two potential candidate genes (i.e., Bol037115 and Bol042270), which exhibiting upregulation after inoculation in the R pool but downregulation in the S pool, were identified from the three QTLs on C07. A functional marker "SWU-OA" was developed from qCRc7-4 on C07, exhibiting ∼95% accuracy in identifying CR in 56 F2 lines. Our study will provide valuable information on resistance genes against P. brassicae and may accelerate the breeding process of B. oleracea with CR.

Silicon Alleviates the Disease Severity of Sclerotinia Stem Rot in Rapeseed.

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is a devastating disease in rapeseed. The objective of this study was to investigate the role and the mechanism of silicon (Si) in alleviating the disease severity of S. sclerotiorum in rapeseed. In the absorption assays, the rapeseed that absorbed 10 mM of K2SiO3 exhibited an 86% decrease in lesion size on infected leaves as compared with controls. In the spray assay, the lesion length on rapeseed stems was reduced by 30.5-32.9% with the use of 100 mM of a foliar Si fertilizer as compared with controls. In the pot assay, the lesion length on rapeseed stems was reduced by 34.9-38.3% when using the Si fertilizer as basal fertilizer. In the field assay, both the disease incidence and disease index of sclerotinia stem rot were significantly reduced with the usage of a solid Si fertilizer, Si foliar fertilizer, and the application of both, without negative affection on the main agronomic traits and seed quality of rapeseed. The transcriptome sequencing, quantitative reverse transcription PCR (qRT-PCR), and biochemical assays between K2SO4- and K2SiO3- treated rapeseed leaves revealed that Si promoted the biosynthesis of defense-related substances and enhanced the antioxidation and detoxification abilities of rapeseed after infection. Thus, this study concluded that Si can alleviate the disease severity of S. sclerotiorum in rapeseeds, partially due to the induced defense responses.

Investigating genetic relationship of Brassica juncea with B. nigra via virtual allopolyploidy and hexaploidy strategy.

Brassica juncea is an important economic crop of the world; however, the narrow genetic base of this crop has tremendously decreased its crop productivity. As an ancestral species of B. juncea, B. nigra is of great importance in widening the genetic diversity of B. juncea. In the present study, 42 SSR markers were employed to screen the genetic diversity among 83 B. nigra, 16 B. juncea, and other Brassica accessions. The molecular characteristics of 498 virtual B. juncea lines were deduced based on the bands of B. nigra and B. rapa via a virtual allopolyploid strategy, and then compared with natural B. juncea accessions. It was found that B. nigra had rich genetic diversity and could be classified into four subgroups, of which subgroup B-III and subgroup B-IV exhibited the closest and the most distant genetic relationship with B. juncea, respectively. To verify this, a hexaploidy strategy was applied to generated synthetic B. juncea from 20 B. nigra accessions, resulting in 45 new-type B. juncea genotypes. The genetic analyses detected that synthetic B. juncea derived from B. nigra in subgroup B-III was close to natural B. juncea, while B. juncea synthesized with B. nigra from subgroup B-IV exhibited wide genetic diversity and was most distant with current B. juncea. This study revealed a great potential of B. nigra in widening genetic diversity of B. juncea particularly using B. nigra in subgroup B-IV, and is helpful in better understanding of the genetic relationship between B. nigra and B. juncea. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-020-01197-7.

Sclerotinia sclerotiorum utilizes host-derived copper for ROS detoxification and infection.

Necrotrophic plant pathogen induces host reactive oxygen species (ROS) production, which leads to necrosis in the host, allowing the pathogen to absorb nutrients from the dead tissues. Sclerotinia sclerotiorum is a typical necrotrophic pathogen that causes Sclerotinia stem rot in more than 400 species, resulting in serious economic losses. Here, we found that three S. sclerotiorum genes involved in copper ion import/transport, SsCTR1, SsCCS and SsATX1, were significantly up-regulated during infection of Brassica oleracea. Function analysis revealed that these genes involved in fungal ROS detoxification and virulence. On the host side, four genes putatively involved in copper ion homeostasis, BolCCS, BolCCH, BolMT2A and BolDRT112, were significantly down-regulated in susceptible B. oleracea, but stably expressed in resistant B. oleracea during infection. Their homologs were found to promote resistance to S. sclerotiorum and increase antioxidant activity in Arabidopsis thaliana. Furthermore, copper concentration analysis indicated that copper flow from healthy area into the necrotic area during infection. A model was proposed that S. sclerotiorum utilizes host copper to detoxify ROS in its cells, whereas the resistant hosts may restrict the supply of essential copper nutrients to S. sclerotiorum by maintaining copper ion homeostasis during infection.

Introgression and pyramiding of genetic loci from wild Brassica oleracea into B. napus for improving Sclerotinia resistance of rapeseed.

KEY MESSAGE: Resistant rapeseed lines pyramided with multiple resistant QTLs derived from Brassica oleracea were developed via a hexaploidy strategy. Rapeseed (Brassica napus L.) suffers heavily from Sclerotinia stem rot, but the breeding of Sclerotinia-resistant rapeseed cultivar has been unsuccessful. During the study, interspecific hexaploids were generated between rapeseed variety 'Zhongshuang 9' and a wild B. oleracea which was highly resistant to S. sclerotiorum, followed by backcrossing with Zhongshuang 9 and successive selfing. By molecular marker-assisted selection, three major resistant QTLs were transferred and pyramided from B. oleracea into two BC1F8 lines which exhibited ~ 35% higher resistance level than Zhongshuang 9 and produced good seed yield and seed quality. It is the first report on successful development of Sclerotinia-resistant rapeseed lines by introducing multiple resistant loci from wild B. oleracea. This study revealed the effectiveness of pyramiding multiple QTLs in improving Sclerotinia resistance in rapeseed and provided a novel breeding strategy on utilization of B. oleracea in rapeseed improvement.

Understanding the Resistance Mechanism in Brassica napus to Clubroot Caused by Plasmodiophora brassicae.

Exploring the mechanism of plant resistance has become the basis for selection of resistance varieties but reports on revealing resistant mechanism in Brassica napus against Plasmodiophora brassicae are rare. In this study, RNA-seq was conducted in the clubroot-resistant B. napus breeding line ZHE-226 and in the clubroot-susceptible rapeseed cultivar Zhongshuang 11 at 0, 3, 6, 9, and 12 days after inoculation. Strong alteration was detected specifically in ZHE-226 as soon as the root hair infection happened, and significant promotion was found in ZHE-226 on cell division or cell cycle, DNA repair and synthesis, protein synthesis, signaling, antioxidation, and secondary metabolites. Combining results from physiological, biochemical, and histochemical assays, our study highlights an effective signaling in ZHE-226 in response to P. brassicae. This response consists of a fast initiation of receptor kinases by P. brassicae; the possible activation of host intercellular G proteins which might, together with an enhanced Ca2+ signaling, promote the production of reactive oxygen species; and programmed cell death in the host. Meanwhile, a strong ability to maintain homeostasis of auxin and cytokinin in ZHE-226 might effectively limit the formation of clubs on host roots. Our study provides initial insights into resistance mechanism in rapeseed to P. brassicae.

Simultaneous Transcriptome Analysis of Host and Pathogen Highlights the Interaction Between Brassica oleracea and Sclerotinia sclerotiorum.

White mold disease caused by Sclerotinia sclerotiorum is a devastating disease of Brassica crops. Here, we simultaneously assessed the transcriptome changes from lesions produced by S. sclerotiorum on disease-resistant (R) and -susceptible (S) B. oleracea pools bulked from a resistance-segregating F2 population. Virulence genes of S. sclerotiorum, including polygalacturonans, chitin synthase, secretory proteins, and oxalic acid biosynthesis, were significantly repressed in lesions of R B. oleracea at 12 h postinoculation (hpi) but exhibited similar expression patterns in R and S B. oleracea at 24 hpi. Resistant B. oleracea induced expression of receptors potentially to perceive Sclerotinia signals during 0 to 12 hpi and deployed complex strategies to suppress the pathogen establishment, including the quick accumulation of reactive oxygen species via activating Ca2+ signaling and suppressing pathogen oxalic acid generation in S. sclerotiorum. In addition, cell wall degradation was inhibited in the resistant B. oleracea potentially to prevent the expansion of Sclerotinia hyphae. The transcriptome changes in S. sclerotiorum and host revealed that resistant B. oleracea produces strong responses against S. sclerotiorum during early infection.

Genome-wide identification of loci affecting seed glucosinolate contents in Brassica napus L.

Glucosinolates are amino acid-derived secondary metabolites that act as chemical defense agents against pests. However, the presence of high levels of glucosinolates severely diminishes the nutritional value of seed meals made from rapeseed (Brassica napus L.). To identify the loci affecting seed glucosinolate content (SGC), we conducted genome-wide resequencing in a population of 307 diverse B. napus accessions from the three B. napus ecotype groups, namely, spring, winter, and semi-winter. These resequencing data were used for a genome-wide association study (GWAS) to identify the loci affecting SGC. In the three ecotype groups, four common and four ecotype-specific haplotype blocks (HBs) were significantly associated with SGC. To identify candidate genes controlling SGC, transcriptome analysis was carried out in 36 accessions showing extreme SGC values. Analyses of haplotypes, genomic variation, and candidate gene expression pointed to five and three candidate genes in the common and spring group-specific HBs, respectively. Our expression analyses demonstrated that additive effects of the three candidate genes in the spring group-specific HB play important roles in the SGC of B. napus.

Genetic characterization and fine mapping for multi-inflorescence in Brassica napus L.

KEY MESSAGE: A major QTL for multi-inflorescence was mapped to a 27.18-kb region on A05 in Brassica napus by integrating QTL mapping, microarray analysis and whole-genome sequencing. Multi-inflorescence is a desirable trait for the genetic improvement of rapeseed (Brassica napus L.). However, the genetic mechanism underlying the multi-inflorescence trait is not well understood. In the present study, a doubled haploid (DH) population derived from a cross between single- and multi-inflorescence lines was investigated for the penetrance of multi-inflorescence across 3 years and genotyped with 257 simple sequence repeat and sequence-related amplified polymorphism loci. A major quantitative trait locus (QTL) for penetrance of multi-inflorescence was mapped to a 9.31-Mb region on chromosome A05, explaining 45.81% of phenotypic variance on average. Subsequently, 13 single-inflorescence and 15 multi-inflorescence DH lines were genotyped with the Brassica microarray, and the QTL interval of multi-inflorescence was narrowed to a 0.74-Mb region with 37 successive single nucleotide polymorphisms between single- and multi-inflorescence groups. A 27.18-kb QTL interval was detected by screening 420 recessive F2 individuals with genome-specific markers. These results will be valuable for gene cloning and molecular breeding of multi-inflorescence in rapeseed.

Identification of Quantitative Trait Loci for Clubroot Resistance in Brassica oleracea With the Use of Brassica SNP Microarray.

Increasing clubroot resistance (CR) of Brassica oleracea by ascertaining the molecular mechanisms has been the key focus in modern B. oleracea breeding. In order to identify the quantitative trait loci (QTLs) associated with CR in B. oleracea, 94 F2 vegetative lines which were developed by tissue culture of selfed seeds from the F1 generation between a clubroot-resistant B. oleracea inbred line and a susceptible line, were identified for disease incidence and six CR-associated traits under a lab inoculation by Plasmodiophora brassicae and were genotyped with the 60K Brassica SNP array. Significant correlations were detected for numbers of fibrous roots and P. brassicae content in roots with disease incidence. Nine linkage groups were constructed from 565 bins which covered around 3,000 SNPs, spanning 1,028 cM of the B. oleracea genome with an average distance of 1.82 cM between adjacent bins. A total of 23 QTLs were identified for disease incidence and the other two correlated traits, individually explaining 6.1-17.8% of the phenotypic variation. Several overlaps were detected among traits, including one three-traits-overlapped locus on linkage group C08 and two important overlapped regions between the two CR-associated traits on C06. The QTLs were compared with known CR loci/genes and the novelty of our QTLs was discussed.

Mapping of genetic locus for leaf trichome in Brassica oleracea.

KEY MESSAGE: The genetic locus for leaf trichome was identified via marker-based mapping and SNP microarray assay, and a functional marker was developed to facilitate the breeding for hairiness in Brassica oleracea. Plant trichomes are involved in various functions particularly in protecting plants against some biotic and abiotic damages. In the present study, an F2 segregating population was developed from the cross between a glabrous cultivated B. oleracea (CC, 2n = 18) and a hairy wild relative, B. incana (CC, 2n = 18). A 1:3 segregation pattern between glabrous and hairy plants was detected among 1063 F2 genotypes, and the locus for hairiness was mapped in a 4.3-cM genetic region using 267 SSR markers among 149 F2 genotypes, corresponding to a 17.6-Mb genomic region on chromosome C01. To narrow the genetic region for hairiness, the Brassica 60 K SNP Bead Chip Arrays were applied to genotype 64 glabrous and 30 hairy F2 plants, resulting in a 1.04-Mb single peak region located in the 17.6-Mb interval. A candidate gene, BoTRY, was identified by qRT-PCR which revealed significant higher expression in glabrous F2 genotypes as compared with that in hairy plants. A cleaved amplified polymorphic site marker was successfully developed to distinguish the sequence variations of BoTRY between hairy and glabrous plants. Our study will be helpful for molecular breeding for hairiness in B. oleracea.

Ss-Rhs1, a secretory Rhs repeat-containing protein, is required for the virulence of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with a worldwide distribution. Cell wall-degrading enzymes and oxalic acid are important to the virulence of this pathogen. Here, we report a novel secretory protein, Ss-Rhs1, which is essential for the virulence of S. sclerotiorum. Ss-Rhs1 is believed to contain a typical signal peptide at the N-terminal and eight rearrangement hotspot (Rhs) repeats. Ss-Rhs1 exhibited a high level of expression at the initial stage of sclerotial development, as well as during the hyphal infection process. Targeted silencing of Ss-Rhs1 resulted in abnormal colony morphology and reduced virulence on host plants. Microscopic observations indicated that Ss-Rhs1-silenced strains exhibited reduced efficiency in compound appressoria formation.

Transcriptomic comparison between Brassica oleracea and rice (Oryza sativa) reveals diverse modulations on cell death in response to Sclerotinia sclerotiorum.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is a devastating disease of Brassica crops, but not in rice. The leaves of a rice line, a partial resistant (R) and a susceptible (S) Brassica oleracea pool that bulked from a resistance-segregating F2 population were employed for transcriptome sequencing before and after inoculation by S. sclerotiorum for 6 and 12 h. Distinct transcriptome profiles were revealed between B. oleracea and rice in response to S. sclerotiorum. Enrichment analyses of GO and KEGG indicated an enhancement of antioxidant activity in the R B. oleracea and rice, and histochemical staining exhibited obvious lighter reactive oxygen species (ROS) accumulation and cell death in rice and the R B. oleracea as compared to that in the S B. oleracea. Significant enhancement of Ca(2+) signalling, a positive regulator of ROS and cell death, were detected in S B. oleracea after inoculation, while it was significantly repressed in the R B. oleracea group. Obvious difference was detected between two B. oleracea groups for WRKY transcription factors, particularly for those regulating cell death. These findings suggest diverse modulations on cell death in host in response to S. sclerotiorum. Our study provides useful insight into the resistant mechanism to S. sclerotiorum.

Time-Series Analyses of Transcriptomes and Proteomes Reveal Molecular Networks Underlying Oil Accumulation in Canola.

Understanding the regulation of lipid metabolism is vital for genetic engineering of canola (Brassica napus L.) to increase oil yield or modify oil composition. We conducted time-series analyses of transcriptomes and proteomes to uncover the molecular networks associated with oil accumulation and dynamic changes in these networks in canola. The expression levels of genes and proteins were measured at 2, 4, 6, and 8 weeks after pollination (WAP). Our results show that the biosynthesis of fatty acids is a dominant cellular process from 2 to 6 WAP, while the degradation mainly happens after 6 WAP. We found that genes in almost every node of fatty acid synthesis pathway were significantly up-regulated during oil accumulation. Moreover, significant expression changes of two genes, acetyl-CoA carboxylase and acyl-ACP desaturase, were detected on both transcriptomic and proteomic levels. We confirmed the temporal expression patterns revealed by the transcriptomic analyses using quantitative real-time PCR experiments. The gene set association analysis show that the biosynthesis of fatty acids and unsaturated fatty acids are the most significant biological processes from 2-4 WAP and 4-6 WAP, respectively, which is consistent with the results of time-series analyses. These results not only provide insight into the mechanisms underlying lipid metabolism, but also reveal novel candidate genes that are worth further investigation for their values in the genetic engineering of canola.

Comparative quantitative trait loci for silique length and seed weight in Brassica napus.

Silique length (SL) and seed weight (SW) are important yield-associated traits in rapeseed (Brassica napus). Although many quantitative trait loci (QTL) for SL and SW have been identified in B. napus, comparative analysis for those QTL is seldom performed. In the present study, 20 and 21 QTL for SL and SW were identified in doubled haploid (DH) and DH-derived reconstructed F2 populations in rapeseed, explaining 55.1-74.3% and 24.4-62.9% of the phenotypic variation across three years, respectively. Of which, 17 QTL with partially or completely overlapped confidence interval on chromosome A09, were homologous with two overlapped QTL on chromosome C08 by aligning QTL confidence intervals with the reference genomes of Brassica crops. By high density selective genotyping of DH lines with extreme phenotypes, using a Brassica single-nucleotide polymorphism (SNP) array, the QTL on chromosome A09 was narrowed, and aligned into 1.14-Mb region from 30.84 to 31.98 Mb on chromosome R09 of B. rapa and 1.05-Mb region from 27.21 to 28.26 Mb on chromosome A09 of B. napus. The alignment of QTL with Brassica reference genomes revealed homologous QTL on A09 and C08 for SL. The narrowed QTL region provides clues for gene cloning and breeding cultivars by marker-assisted selection.

Development of genic cleavage markers in association with seed glucosinolate content in canola.

KEY MESSAGE: The orthologues of Arabidopsis involved in seed glucosinolates metabolism within QTL confidence intervals were identified, and functional markers were developed to facilitate breeding for ultra-low glucosinolates in canola. Further reducing the content of seed glucosinolates will have a positive impact on the seed quality of canola (Brassica napus). In this study 43 quantitative trait loci (QTL) for seed glucosinolate (GSL) content in a low-GSL genetic background were mapped over seven environments in Germany and China in a doubled haploid population from a cross between two low-GSL oilseed rape parents with transgressive segregation. By anchoring these QTL to the reference genomes of B. rapa and B. oleracea, we identified 23 orthologues of Arabidopsis involved in GSL metabolism within the QTL confidence intervals. Sequence polymorphisms between the corresponding coding regions of the parental lines were used to develop cleaved amplified polymorphic site markers for two QTL-linked genes, ISOPROPYLMALATE DEHYDROGENASE1 and ADENOSINE 5'-PHOSPHOSULFATE REDUCTASE 3. The genic cleavage markers were mapped in the DH population into the corresponding intervals of QTL explaining 3.36-6.88 and 4.55-8.67 % of the phenotypic variation for seed GSL, respectively. The markers will facilitate breeding for ultra-low seed GSL content in canola.